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PubMedDate not supplied

Increasing the acetyl-CoA pool in the presence of overexpressed phosphoenolpyruvate carboxylase or pyruvate carboxylase enhances succinate production in Escherichia coli.

An in vivo strategy to apply the activation effect of acetyl-CoA on phosphoenolpyruvate carboxylase (PEPC) and pyruvate carboxylase (PYC) to increase succinate production in Escherichia coli was studied. This approach relies on the increased intracellular acetyl-CoA and CoA levels by overexpressing E. coli pantothenate kinase (PANK). The results showed that coexpression of PANK and PEPC, and PANK and PYC, did improve succinate production compared to the individual expression of PEPC and PYC, respectively. The intracellular acetyl-CoA and CoA levels were also measured, and each showed a significant increase when the PANK was overexpressed. Another effect observed was a decrease in lactate production. The least amount of lactate was produced when PANK and PEPC, and PANK and PYC, were coexpressed. This result showed increased competitiveness of the succinate pathway at the phosphoenolpyruvate and pyruvate nodes for the carbon flux, as a result reducing the carbon flux toward the lactate pathway. The study also demonstrates a feasible method for metabolic engineering to modulate enzyme activity in vivo through specific activators and inhibitors.

Acetyl Coenzyme A↗
PubMed1992

A quantitative immunoassay using chicken antibodies for detection of native and recombinant alpha-amidating enzyme.

A sensitive competitive ELISA has been developed for the detection and quantitation of native and recombinant alpha-amidating enzyme. Chickens immunized with purified enzyme (75 kDa) isolated from a rat medullary thyroid carcinoma, produced IgY antibodies specific for the native enzyme. The assay is defined by a standard curve with a linear range of 0.78-12.5 ng/ml in phosphate-buffered saline, and a limit of sensitivity for detection of the enzyme of 0.20 ng/ml. The immunoassay is capable of detecting enzyme from both tumor derived sources, and from cells genetically engineered to secrete the enzyme into tissue culture medium containing up to 10% fetal calf serum.

Animals↗
PubMed2005

Expanding the alkane oxygenase toolbox: new enzymes and applications.

As highly reduced hydrocarbons are abundant in the environment, enzymes that catalyze the terminal or subterminal oxygenation of alkanes are relatively easy to find. A number of these enzymes have been biochemically characterized in detail, because the potential of alkane hydroxylases to catalyze high added-value reactions is widely recognized. Nevertheless, the industrial application of these enzymes is restricted owing to the complex biochemistry, challenging process requirements, and the limited number of cloned and expressed enzymes. Rational and evolutionary engineering approaches have started to yield more robust and versatile enzyme systems, broadening the alkane oxygenase portfolio. In addition, metagenomic approaches provide access to many novel alkane oxygenase sequences.

Alkanes↗
PubMedDate not supplied

Biotechnological methods to accelerate cheddar cheese ripening.

Cheese is one of the dairy products that can result from the enzymatic coagulation of milk. The basic steps of the transformation of milk into cheese are coagulation, draining, and ripening. Ripening is the complex process required for the development of a cheese's flavor, texture and aroma. Proteolysis, lipolysis and glycolysis are the three main biochemical reactions that are responsible for the basic changes during the maturation period. As ripening is a relatively expensive process for the cheese industry, reducing maturation time without destroying the quality of the ripened cheese has economic and technological benefits. Elevated ripening temperatures, addition of enzymes, addition of cheese slurry, attenuated starters, adjunct cultures, genetically engineered starters and recombinant enzymes and microencapsulation of ripening enzymes are traditional and modern methods used to accelerate cheese ripening. In this context, an up to date review of Cheddar cheese ripening is presented.

Biotechnology↗
PubMed2006

Structural basis for regioselectivity and stereoselectivity of product formation by naphthalene 1,2-dioxygenase.

Rieske oxygenase (RO) systems are two- and three-component enzyme systems that catalyze the formation of cis-dihydrodiols from aromatic substrates. Degradation of pollutants in contaminated soil and generation of chiral synthons have been the major foci of RO research. Substrate specificity and product regio- and stereoselectivity have been shown to vary between individual ROs. While directed evolution methods for altering RO function have been successful in the past, rational engineering of these enzymes still poses a challenge due to the lack of structural understanding. Here we examine the structural changes induced by mutation of Phe-352 in naphthalene 1,2-dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NDO-O(9816-4)). Structures of the Phe-352-Val mutant in native form and in complex with phenanthrene and anthracene, along with those of wild-type NDO-O(9816-4) in complex with phenanthrene, anthracene, and 3-nitrotoluene, are presented. Phenanthrene was shown to bind in a different orientation in the Phe-352-Val mutant active site from that in the wild type, while anthracene was found to bind in similar positions in both enzymes. Two orientations of 3-nitrotoluene were observed, i.e., a productive and a nonproductive orientation. These orientations help explain why NDO-O(9816-4) forms different products from 3-nitrotoluene than those made from nitrobenzene dioxygenase. Comparison of these structures among themselves and with other known ROs bound to substrates reveals that the orientation of substrate binding at the active site is the primary determinant of product regio- and stereoselectivity.

Anthracenes↗
PubMed1998

A competitive immunoassay for the detection of esterolytic activity of antibodies and enzymes.

Screening of a large number of clones produced in a fusion is often the bottleneck in the isolation of catalytic antibodies. The usual approach requires two steps: clones are first selected for their high affinity to the antigen, and then the good binders are tested for their catalytic activity. To simplify this selection process, a competitive enzyme-linked immunosorbent assay (ELISA) has been developed that allows direct screening of the antibodies on the basis of their catalytic activity. In this assay, the product of the catalyzed reaction binds to an immobilized anti-product antibody in competition with a peroxidase-product conjugate. The screening assay has been developed for the antibody-catalyzed hydrolysis of esters of p-aminophenylacetic acid and has been tested on the porcine liver esterase (PLE)-catalyzed hydrolysis of the same substrates. This test allows the detection of product formation at the nanomolar level, while, in a typical assay, the catalytic activity of PLE can be traced down to 200 fmol of enzyme. Under standard conditions for the screening of hybridomas obtained from a fusion, the competitive ELISA allows detection of catalytic species with values of kcat > or = 5 x 10(-7) mol l-1 s-1 and kcat/kuncat > or = 50. While the assay has been designed for the selection of catalytic antibodies, other potential applications of this methodology are in the screening of libraries of engineered and designed enzymes and, in general, in the quantitative measurement of enzyme activity.

Animals↗
PubMed1998

T4 DNA ligase synthesizes dinucleoside polyphosphates.

T4 DNA ligase (EC 6.5.1.1), one of the most widely used enzymes in genetic engineering, transfers AMP from the E-AMP complex to tripolyphosphate, ADP, ATP, GTP or dATP producing p4A, Ap3A, Ap4A, Ap4G and Ap4dA, respectively. Nicked DNA competes very effectively with GTP for the synthesis of Ap4G and, conversely, tripolyphosphate (or GTP) inhibits the ligation of DNA by the ligase. As T4 DNA ligase has similar requirements for ATP as the mammalian DNA ligase(s), the latter enzyme(s) could also synthesize dinucleoside polyphosphates. The present report may be related to the recent finding that human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5',5''-P1,P3-triphosphate (Ap3A) hydrolase (EC 3.6.1.29).

Acid Anhydride Hydrolases↗
PubMed2001

Engineering the CYP101 system for in vivo oxidation of unnatural substrates.

The protein engineering of CYP enzymes for structure-activity studies and the oxidation of unnatural substrates for biotechnological applications will be greatly facilitated by the availability of functional, whole-cell systems for substrate oxidation. We report the construction of a tricistronic plasmid that expresses the CYP101 monooxygenase from Pseudomonas putida, and its physiological electron transfer co-factor proteins putidaredoxin reductase and putidaredoxin in Escherichia coli, giving a functional in vivo catalytic system. Wild-type CYP101 expressed in this system efficiently transforms camphor to 5-exo-hydroxycamphor without further oxidation to 5-oxo-camphor until >95% of camphor has been consumed. CYP101 mutants with increased activity for the oxidation of diphenylmethane (the Y96F-I395G mutant), styrene and ethylbenzene (the Y96F-V247L mutant) have been engineered. In particular, the Y96F-V247L mutant shows coupling efficiency of approximately 60% for styrene and ethylbenzene oxidation, with substrate oxidation rates of approximately 100/min. Escherichia coli cells transformed with tricistronic plasmids expressing these mutants readily gave 100-mg quantities of 4-hydroxydiphenylmethane and 1-phenylethanol in 24-72 h. This new in vivo system can be used for preparative scale reactions for product characterization, and will greatly facilitate directed evolution of the CYP101 enzyme for enhanced activity and selectivity of substrate oxidation.

Alkanes↗
PubMed2007

Growth of the genetically engineered strain Cupriavidus necator RW112 with chlorobenzoates and technical chlorobiphenyls.

Cupriavidus necator (formerly Ralstonia eutropha) strain H850 is known to grow on biphenyl, and to co-oxidize congeners of polychlorinated biphenyls (PCBs). Using a Tn5-based minitransposon shuttle system and the TOL plasmid, the rational construction of hybrids of H850 was achieved by subsequent introduction of three distinct elements carrying 11 catabolic loci from three other biodegrading bacteria into the parent strain, finally yielding C. necator RW112. The new genetic elements introduced into H850 and its derivatives were tcbRCDEF, which encode the catabolic enzymes needed for chlorocatechol biodegradation under the control of a transcriptional regulator, followed by cbdABC, encoding a 2-halobenzoate dioxygenase, and xylXYZ, encoding a broad-spectrum toluate dioxygenase. The expression of the introduced genes was demonstrated by measuring the corresponding enzymic activities. The engineered strain RW112 gained the ability to grow on all isomeric monochlorobenzoates and 3,5-dichlorobenzoate, all monochlorobiphenyls, and 3,5-dichloro-, 2,3'-dichloro- and 2,4'-dichlorobiphenyl, without accumulation of chlorobenzoates. It also grew and utilized two commercial PCB formulations, Aroclor 1221 and Aroclor 1232, as sole carbon and energy sources for growth. This is the first report on the aerobic growth of a genetically improved bacterial strain at the expense of technical Aroclor mixtures.

Aroclors↗
PubMed2004

Transcription factors as tools for metabolic engineering in plants.

The functions of an increasing number of plant transcription factors are being elucidated, and many of these factors have been found to impact flux through metabolic pathways. Because transcription factors, as opposed to most structural genes, tend to control multiple pathway steps, they have emerged as powerful tools for the manipulation of complex metabolic pathways in plants. The review describes the highlights of recent experiments that have targeted transcription factors that control plant metabolic pathways, and discusses their potential as tools for metabolic engineering.

Enzymes↗
PubMed2006

Microbial degradation of organophosphorus xenobiotics: metabolic pathways and molecular basis.

Organophosphorus (OP) xenobiotics are used worldwide as pesticides and petroleum additives. OP compounds share the major portion of the pesticide market globally. Owing to large-scale use of OP compounds, contaminations of soil and water systems have been reported from all parts of the world. OP compounds possess very high mammalian toxicity and therefore early detection and subsequent decontamination and detoxification of the polluted environment is essential. Additionally, about 200,000 tons of extremely toxic OP chemical warfare agents are required to be destroyed by 2007 under Chemical Warfare Convention (1993). Chemical and physical methods of decontamination are not only expensive and time-consuming, but also in most cases they do not provide a complete solution. These approaches convert compounds from toxic into less toxic states, which in some cases can accumulate in the environment and still be toxic to a range of organisms. Bioremediation provides a suitable way to remove contaminants from the environment as, in most of the cases, OP compounds are totally mineralized by the microorganisms. Most OP compounds are degraded by microorganisms in the environment as a source of phosphorus or carbon or both. Several soil bacteria have been isolated and characterized, which can degrade OP compounds in laboratory cultures and in the field. The biochemical and genetic basis of microbial degradation has received considerable attention. Several genes/enzymes, which provide microorganisms with the ability to degrade OP compounds, have been identified and characterized. Some of these genes and enzymes have been engineered for better efficacy. Bacteria capable of complete mineralization are constructed by transferring the complete degradation pathway for specific compounds to one bacterium. In the present article, we review microbial degradation and metabolic pathways for some OP compounds. The biochemical and molecular basis of OP degradation by microbes and the evolution and distribution of genes/enzymes are also reviewed. This article also examines applications and future use of OP-degrading microbes and enzymes for bioremediation, treatment of OP poisoning, and as biosensors.

Aryldialkylphosphatase↗
PubMed2002

Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes.

The accumulation of compatible solutes, such as betaines, proline and sugar alcohols, is a widespread response that may protect plants against environmental stress. It is not yet fully understood how these compounds are involved in the stress tolerance of whole plants. Some plants have been genetically engineered to express enzymes that catalyze the synthesis of various compatible solutes. Some interventions have increased the tolerance of some crop plants to abiotic stress. Furthermore, analysis of such transgenic plants has begun to clarify the roles of compatible solutes in stress tolerance.

Adaptation, Physiological↗
PubMed2004

BRENDA, the enzyme database: updates and major new developments.

BRENDA (BRaunschweig ENzyme DAtabase) represents a comprehensive collection of enzyme and metabolic information, based on primary literature. The database contains data from at least 83,000 different enzymes from 9800 different organisms, classified in approximately 4200 EC numbers. BRENDA includes biochemical and molecular information on classification and nomenclature, reaction and specificity, functional parameters, occurrence, enzyme structure, application, engineering, stability, disease, isolation and preparation, links and literature references. The data are extracted and evaluated from approximately 46,000 references, which are linked to PubMed as long as the reference is cited in PubMed. In the past year BRENDA has undergone major changes including a large increase in updating speed with >50% of all data updated in 2002 or in the first half of 2003, the development of a new EC-tree browser, a taxonomy-tree browser, a chemical substructure search engine for ligand structure, the development of controlled vocabulary, an ontology for some information fields and a thesaurus for ligand names. The database is accessible free of charge to the academic community at http://www.brenda. uni-koeln.de.

Animals↗
PubMed1996

Mutagenesis study of Asp-290 in cytochrome P450 2B11 using a fusion protein with rat NADPH-cytochrome P450 reductase.

Asp-290 of the phenobarbital-inducible dog liver cytochrome P450 (P450) 2B11 was mutated to nine other amino acid residues by site-directed mutagenesis, and the functional significance of the unique negative charge in P450 2B11 at that position was studied. To facilitate the analysis of mutated P450 2B11 enzymes heterologously expressed in Escherichia coli, an enzymatically active fusion enzyme was genetically engineered between the cDNAs for P450 2B11 and rat liver NADPH-cytochrome P450 reductase using a Ser-Thr linker as previously described (Fisher et al., 1992, Proc. Natl. Acad. Sci. USA 89, 10817-10821). Sonicated whole-cell lysates of E. coli cells expressing the wild-type fusion protein were able to catalyze the 16-hydroxylation of androstenedione (AD) in the absence of added reductase, and exhibited activities and androstenedione metabolite profiles very similar to those of purified and reconstituted enzyme preparations. The substitution of Ala, Glu, Gly, Met, Asn, Arg, Ser, Thr, or Val for Asp-290 of P450 2B11 resulted in decreased AD hydroxylase activities as assessed using solubilized membranes. Replacement of Asp-290 with Glu yielded the highest activity (55% of wild type), while substituting the positively charged amino acid Arg created an enzyme with the lowest activity (< 1% of wild-type activity). Regioselectivity of AD hydroxylation was not affected although the stereoselectivity of hydroxylation at the 16 carbon position was altered in some cases. The use of the fused enzyme to study the effects of site-directed mutagenesis has resulted in the demonstration of the importance of size and charge at position 290 for enzymatic activity of P450 2B11.

Animals↗
PubMed1991

Mechanism of mutual activation of the tryptophan synthase alpha and beta subunits. Analysis of the reaction specificity and substrate-induced inactivation of active site and tunnel mutants of the beta subunit.

The origin of reaction and substrate specificity and the control of activity by protein-protein interaction are investigated using the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. We have compared some spectroscopic and kinetic properties of the wild type beta subunit and five mutant forms of the beta subunit that have altered catalytic properties. These mutant enzymes, which were engineered by site-directed mutagenesis, have single amino acid replacements in either the active site or in the wall of a tunnel that extends from the active site of the alpha subunit to the active site of the beta subunit in the alpha 2 beta 2 complex. We find that the mutant alpha 2 beta 2 complexes have altered reaction and substrate specificity in beta-elimination and beta-replacement reactions with L-serine and with beta-chloro-L-alanine. Moreover, the mutant enzymes, unlike the wild type alpha 2 beta 2 complex, undergo irreversible substrate-induced inactivation. The mechanism of inactivation appears to be analogous to that first demonstrated by Metzler's group for inhibition of two other pyridoxal phosphate enzymes. Alkaline treatment of the inactivated enzyme yields apoenzyme and a previously described pyridoxal phosphate derivative. We demonstrate for the first time that enzymatic activity can be recovered by addition of pyridoxal phosphate following alkaline treatment. We conclude that the wild type and mutant alpha 2 beta 2 complexes differ in the way they process the amino acrylate intermediate. We suggest that the wild type beta subunit undergoes a conformational change upon association with the alpha subunit that alters the reaction specificity and that the mutant beta subunits do not undergo the same conformational change upon subunit association.

Base Sequence↗
PubMed2004

Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity.

Amylosucrase is a glucosyltransferase belonging to family 13 of glycoside hydrolases and catalyses the formation of an amylose-type polymer from sucrose. Its potential use as an industrial tool for the synthesis or the modification of polysaccharides, however, is limited by its low catalytic efficiency on sucrose alone, its low stability, and its side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling, and selective screening (directed evolution) was started, in order to generate more efficient variants of the enzyme. A convenient zero background expression cloning strategy was developed. Mutant gene libraries were generated by error-prone polymerase chain reaction (PCR), using Taq polymerase with unbalanced dNTPs or Mutazyme trade mark, followed by recombination of the PCR products by DNA shuffling. A selection method was developed to allow only the growth of amylosucrase active clones on solid mineral medium containing sucrose as the sole carbon source. Automated protocols were designed to screen amylosucrase activity from mini-cultures using dinitrosalicylic acid staining of reducing sugars and iodine staining of amylose-like polymer. A pilot experiment using the described mutagenesis, selection, and screening methods yielded two variants with significantly increased activity (five-fold under the screening conditions). Sequence analysis of these variants revealed mutations in amino acid residues which would not be considered for rational design of improved amylosucrase variants. A method for the characterisation of amylosucrase action on sucrose, consisting of accurate measurement of glucose and fructose concentrations, was introduced. This allows discrimination between hydrolysis and transglucosylation, enabling a more detailed comparison between wild-type and mutant enzymes.

Catalysis↗